1. Field of the invention
This invention relates to peptides useful in metalloproteinase detection and inhibition. Specifically, the invention relates to peptides derived from the sequence of type IV collagenase which correspond to domain of the enzyme involved in enzyme activation and interaction of the enzyme with the substrate. Antibodies recognizing the peptides are useful in enzyme detection. Specific peptides, identified based on functional studies, constitute new classes of metalloproteinase inhibitors.
2. Background
The degradation of interstitial and basement membrane collagens is initiated by a specific class of metalloproteinases, the collagenases (EC 3.4.24.7), which are secreted into the extracellular matrix in zymogen form. The interstitial collagenases which degrade collagen types I, II and III have been characterized with respect to substrate specificity and requirements for activation (1-5).
Pepsinized type IV collagen is not susceptible to degradation by these enzymes, but is instead degraded in a specific fashion by an enzyme that has been identified in human tumor cells (6,7,11,14,17), endothelial cells (8), bone (24), fibroblasts (17), polymorphonuclear leukocytes (9) and macrophages (10). This enzyme, referred to as type IV collagenase (11,17) is a neutral metalloproteinase of 68 to 72 kilodaltons which is secreted in zymogen form (11-13). This enzyme has been closely linked to the metastatic potential of tumors in murine tumor models (14) and is augmented following the H-ras oncogene induced genetic induction of the metastatic phenotype (15,16). Trypsin treatment results in activation of the latent enzyme and a concomitant reduction in the molecular mass (12). Organomercurial compounds have also been shown to activate this enzyme, and these are also associated with a reduction in the molecular mass (17,24). The activated enzyme cleaves type IV collagen to generate characteristic 1/4 amino-terminal and 3/4 carboxy-terminal fragments (12,17,18). It has also been demonstrated that gelatinolytic activity is associated with this enzyme (17,19,24) as well as a type V collagenolytic activity (17,24).
Type IV collagenase has been purified from human melanoma cells and sequence information on the intact protein amino terminus has been obtained as well as on tryptic and cyanogen bromide peptide fragments (19). The sequence information demonstrates that type IV collagenase shows limited sequence homology to interstitial collagenase and stromelysin. A recent report has characterized a partial cDNA clone for a metalloproteinase secreted by H-ras-transformed human bronchial epithelial cells (17). The transformed bronchial epithelial enzyme is capable of specifically degrading type IV collagen, and the deduced amino acid sequence shows identity with that reported for tryptic and cyanogen bromide fragments of human tumor type IV collagenase (19). Thus, human melanoma cell type IV collagenase appears identical with the enzyme from H-ras-transformed bronchial epithelial cells, which is also found in fibroblasts (17) and bone cell explants (24).